Affinity For Histamine Research Articles

Pharmacological Characterization of the Zebrafish (Danio Rerio) Histamine H1 Receptor Reveals the Involvement of the Second Extracellular Loop in the Binding of Histamine.

The zebrafish (Danio rerio) histamine H1 receptor gene (zfH1R) was cloned in 2007 and reported to be involved in fish locomotion. Yet, no detailed characterization of its pharmacology and signaling properties have so far been reported. In this study, we pharmacologically characterized the zfH1R expressed in HEK-293T cells by means of [3H]-mepyramine binding and G protein-signaling assays. The zfH1R [dissociation constant (KD), 0.7 nM] displayed similar affinity for the antagonist [3H]-mepyramine as the human histamine H1 receptor (hH1R) (KD, 1.5 nM), whereas the affinity for histamine is 100-fold higher than for the human H1R. The zfH1R couples to Gαq/11 proteins and activates several reporter genes, i.e., NFAT, NFϰB, CRE, VEGF, COX-2, SRE, and AP-1, and zfH1R-mediated signaling is prevented by the Gαq/11 inhibitor YM-254890 and the antagonist mepyramine. Molecular modeling of the zfH1R and human H1R shows that the binding pockets are identical, implying that variations along the ligand binding pathway could underly the differences in histamine affinity instead. Targeting differentially charged residues in extracellular loop 2 (ECL2) using site-directed mutagenesis revealed that Arg21045x55 is most likely involved in the binding process of histamine in zfH1R. This study aids the understanding of the pharmacological differences between H1R orthologs and the role of ECL2 in histamine binding and provides fundamental information for the understanding of the histaminergic system in the zebrafish. SIGNIFICANCE STATEMENT: The use of the zebrafish as in vivo models in neuroscience is growing exponentially, which asks for detailed characterization of the aminergic neurotransmitter systems in this model. This study is the first to pharmacologically characterize the zebrafish histamine H1 receptor after expression in HEK-293T cells. The results show a high pharmacological and functional resemblance with the human ortholog but also reveal interesting structural differences and unveils an important role of the second extracellular loop in histamine binding.

Effect of cross-linking agents on the adsorption of histamine on molecularly imprinted polyacrylamide

The ability of molecularly imprinted polymers (MIPs) prepared with flexible cross-linking agents for selective uptake of guest molecules is investigated. New polyacrylamide polymers had been synthesized for histamine (HA) extraction using solid supports based on MIPs immobilized on silica surface (SiO2). The MIPs were prepared via radical polymerization in acetonitrile and in the presence of azobisisobutyronitrile (AIBN) as initiator, using HA as a target molecule, acrylamide as a functional monomer and several cross-linking agents having alkyl spacers of various lengths: N,N′-methylenebisacrylamide (BA1), N,N′-ethylenebisacrylamide (BA2), N,N’-(butane-1,4-diyl)-bis(2-methacrylamide) (BA4) and N,N’-(hexane-1,6-diyl)-bis(2-methacrylamide) (BA6). All the prepared MIPs have a strong absorption capacity of HA compared to non-imprinted polyacrylamide (NIPs). MIP4 made with the BA4 cross-linker shows a higher selectivity for adsorption of HA than MIPs made with BA1, BA2 and BA6. Thus, the imprinting factor for adsorption from a 5 ppm HA solution was 2.5 for MIP4 against 1.4 to 1.6 for MIP1, MIP2 and MIP6. Langmuir–Volmer and Volmer models of the adsorption isotherms of MIPs and NIPs accurately fit the experimental data. They provide the characteristic parameters of the adsorption thermodynamic of the materials. The higher performance of MIP4 for selective adsorption of HA is mainly coming from a higher density of molecular imprints (1.4 μmol m−2 against 0.13–0.15 μmol m−2 for MIP1 and MIP2 and 0.4 μmol m−2 for MIP6) than a higher affinity of HA for the molecular imprints. The selectivity with respect to the tyramine and melamine interfering molecules is also higher for MIP4. As the main outcome, some moderate flexibility of the material is advantageous. Such flexibility allows for a reversible healing and re-opening of the molecular imprints as the HA guest is removed and back-adsorbed.

Leki przeciwdepresyne typu SNRI — medycyna oparta na faktach i doświadczeniu

Serotonin-norepinephrine reuptake inhibitors (SNRI) antidepressants are drugs registered for the treatment of depression and anxiety disorders. These drugs affect the reuptake of both serotonin and noradrenaline. There are two such drugs available in Poland: venlafaxine and duloxetine. The mechanism of venlafaxine’s antidepressant effect in humans is believed to be related to increased neurotransmitter activity in the central nervous system (CNS). Preclinical studies have shown that venlafaxine and its major metabolite (O-demethylenlafaxine, ODV) are serotonin and norepinephrine reuptake inhibitors. Venlafaxine is also a weak dopamine reuptake inhibitor. Venlafaxine acts by blocking neurotransmitter transport and reuptake proteins and acts through postsynaptic terminals, ultimately enhancing postsynaptic stimulation. Duloxetine is an inhibitor of serotonin (5-HT) and norepinephrine (NA) reuptake. It weakly inhibits dopamine reuptake (e.g. in frontal regions), and has no significant affinity for histamine, dopaminergic, cholinergic and adrenergic receptors. Duloxetine dose-dependently increases extracellular serotonin and norepinephrine concentrations in different brain areas in animals. In this paper we will discuss the most important literature data concerning the above mentioned drugs, as well as define a patient profile that could most benefit from individually selected treatment.

Characteristics of histamine H4 receptor agonist-induced allergic conjunctivitis model in Guinea pigs

Histamine is strongly associated with the onset of allergic conjunctivitis. The most recent cloned histamine H4 receptor antagonist is highly expected as a new therapeutic drug candidate. As a model for a therapeutic drug targeting the histamine H4 receptor, a mouse model in which conjunctivitis symptoms are induced by instilling 4-methylhistamine, a histamine H4 receptor agonist, has been reported. However, the affinity of the H4 receptor for histamine varies in species, and it is known that the histamine binding affinity for the guinea pig H4 receptor is closer to that for human receptor than mice receptor. In this paper, we investigated a possibility that a guinea pig model would become a drug efficacy evaluation model with higher evaluation accuracy than the mouse model.As a result, hyperemia was observed in the conjunctivae and iris of guinea pigs after instillation of 4-methylhistamine and specifically suppressed by the histamine H4 receptor antagonist. Unlikely to the previously reported mouse model, however, none of edema, increased vascular permeability or scratching behavior was observed, suggesting that there may be differences between mice and guinea pigs not only in the binding affinity of histamine to the H4 receptor but also in the biological reaction to 4-methylhistamine. Although the symptoms of allergic conjunctivitis do not appear comprehensively in the guinea pig model, results of this study indicated a possibility that this model can be used as a simple screening model in the early stages of drug development.

Colorimetric sensing of histamine in aqueous solution by a system composed of alizarin complexone and Ni2+ complex via indicator displacement approach

The complex composed of alizarin complexone (ALC) and Ni2+ has been developed as a colorimetric probe for the selective recognition of histamine over other neurotransmitters in an aqueous solution via the indicator displacement approach. ALC formed a complex with Ni2+ in a 1:1 ratio (ALC-Ni2+) accompanied by the color change from red to purple. The detection of histamine is based on high affinity of histamine for Ni2+. The addition of histamine to ALC-Ni2+ caused the complex formation of histamine with Ni2+ in a 1:1 stoichiometry, resulting a hypsochromic shift accompanied by change back of color from purple to red through the release of ALC from ALC-Ni2+. The absorption response is linear with the histamine concentration in the range of 5–150 μM with a detection limit of 7.68 μM. Moreover, other neurotransmitters did not show any significant interference with the histamine detection from the competition experiments. Thus, ALC-Ni2+ can be used as a simple probe for histamine in aqueous solution.

Chapter 23 - Histamine receptors, agonists, and antagonists in health and disease

Histamine in the brain is produced by a group of tuberomamillary neurons in the posterior hypothalamus and a limited number of mast cells in different parts of the brain. Four G-protein-coupled receptors mediate the effects of histamine. Two of these receptors, H3 and H4 receptors, are high-affinity receptors in the brain and immune system, respectively. The two classic histamine receptors, H1 receptor and H2 receptor, are well known as drug targets for allergy and gastric ulcer, respectively. These receptors have lower affinity for histamine than the more recently discovered H3 and H4 receptors. The H1 and H2 receptors are important postsynaptic receptors in the brain, and they mediate many of the central effects of histamine on, e.g., alertness and wakefulness. H3 receptor is a pre- and postsynaptic receptor, which regulates release of histamine and several other neurotransmitters, including serotonin, GABA, and glutamate. H4 receptor is found in cerebral blood vessels and microglia, but its expression in neurons is not yet well established. Pitolisant, a H3 receptor antagonist, is used to treat narcolepsy and hypersomnia. H1 receptor antagonists have been used to treat insomnia, but its use requires precautions due to potential side effects. H2 receptor antagonists have shown efficacy in treatment of schizophrenia, but they are not in widespread clinical use. H4 receptor ligands may in the future be tested for neuroimmunological disorders and potentially neurodegenerative disorders in which inflammation plays a role, but clinical tests have not yet been initiated.

The Effect of Deuteration on the H2 Receptor Histamine Binding Profile: A Computational Insight into Modified Hydrogen Bonding Interactions.

We used a range of computational techniques to reveal an increased histamine affinity for its H2 receptor upon deuteration, which was interpreted through altered hydrogen bonding interactions within the receptor and the aqueous environment preceding the binding. Molecular docking identified the area between third and fifth transmembrane α-helices as the likely binding pocket for several histamine poses, with the most favorable binding energy of −7.4 kcal mol−1 closely matching the experimental value of −5.9 kcal mol−1. The subsequent molecular dynamics simulation and MM-GBSA analysis recognized Asp98 as the most dominant residue, accounting for 40% of the total binding energy, established through a persistent hydrogen bonding with the histamine −NH3+ group, the latter further held in place through the N–H∙∙∙O hydrogen bonding with Tyr250. Unlike earlier literature proposals, the important role of Thr190 is not evident in hydrogen bonds through its −OH group, but rather in the C–H∙∙∙π contacts with the imidazole ring, while its former moiety is constantly engaged in the hydrogen bonding with Asp186. Lastly, quantum-chemical calculations within the receptor cluster model and utilizing the empirical quantization of the ionizable X–H bonds (X = N, O, S), supported the deuteration-induced affinity increase, with the calculated difference in the binding free energy of −0.85 kcal mol−1, being in excellent agreement with an experimental value of −0.75 kcal mol−1, thus confirming the relevance of hydrogen bonding for the H2 receptor activation.

Evolutionary history of histamine receptors: Early vertebrate origin and expansion of the H3-H4 subtypes

Histamine receptors belonging to the superfamily of G protein-coupled receptors (GPCRs) mediate the diverse biological effects of biogenic histamine. They are classified into four phylogenetically distinct subtypes H1-H4, each with a different binding affinity for histamine and divergent downstream signaling pathways. Here we present the evolutionary history of the histamine receptors using a phylogenetic approach complemented with comparative genomics analyses of the sequences, gene structures, and synteny of gene neighborhoods. The data indicate the earliest emergence of histamine-mediated GPCR signaling by a H2 in a prebilaterian ancestor. The analyses support a revised classification of the vertebrate H3-H4 receptor subtypes. We demonstrate the presence of the H4 across vertebrates, contradicting the currently held notion that H4 is restricted to mammals. These non-mammalian vertebrate H4 orthologs have been mistaken for H3. We also identify the presence of a new H3 subtype (H3B), distinct from the canonical H3 (H3A), and propose that the H3A, H3B, and H4 likely emerged from a H3 progenitor through the 1R/2R whole genome duplications in an ancestor of the vertebrates. It is apparent that the ability of the H1, H2, and H3-4 to bind histamine was acquired convergently. We identified genomic signatures suggesting that the H1 and H3-H4 shared a last common ancestor with the muscarinic receptor in a bilaterian predecessor whereas, the H2 and the α-adrenoreceptor shared a progenitor in a prebilaterian ancestor. Furthermore, site-specific analysis of the vertebrate subtypes revealed potential residues that may account for the functional divergence between them.

Zinc(II) salphen complex-based fluorescence optical sensor for biogenic amine detection.

Biogenic amines have attracted interest among researchers because of their importance as biomarkers in determining the quality of food freshness in the food industry. A rapid and simple technique that is able to detect biogenic amines is needed. In this work, a new optical sensing material for one of the biogenic amines, histamine, based on a new zinc(II) salphen complex was developed. The binding of zinc(II) complexes without an electron-withdrawing group (complex 1) and with electron-withdrawing groups (F, complex 2; Cl, complex 3) to histamine resulted in enhancement of fluorescence. All complexes exhibited high affinity for histamine [binding constant of (7.14 ± 0.80) × 104, (3.33 ± 0.03) × 105, and (2.35 ± 0.14) × 105 M-1, respectively]. Complex 2 was chosen as the sensing material for further development of an optical sensor for biogenic amines in the following step since it displayed enhanced optical properties in comparison with complexes 1 and 3. The optical sensor for biogenic amines used silica microparticles as the immobilisation support and histamine as the analyte. The optical sensor had a limit of detection for histamine of 4.4 × 10-12 M, with a linear working range between 1.0 × 10-11 and 1.0 × 10-6 M (R2 = 0.9844). The sensor showed good reproducibility, with a low relative standard deviation (5.5 %). In addition, the sensor exhibited good selectivity towards histamine and cadaverine over other amines, such as 1,2-phenylenediamine, triethylamine, and trimethylamine. Recovery and real sample studies suggested that complex 2 could be a promising biogenic amine optical sensing material that can be applied in the food industry, especially in controlling the safety of food for it to remain fresh and healthy for consumption.

Effect of the Fexofenadine on the expression of HRH-1 and HRH-4 receptor in Peripheral Blood Mononuclear Cell isolated from children with diagnosed allergy - in vitro study Short communication.

Fexofenadine (FXF) is the active metabolite of terfenadine with selective peripheral H1receptor antagonist activity. FXF is a third-generation antihistamine, non-sedating, rapid and very long acting used in symptoms associated with allergic diseases such as allergic rhinitis, asthma and dermatitis. The pleiotropic effects of histamine are mediated by four types of receptors that belong to the G-protein-coupled receptor family: histamine H1 receptor (HRH-1), histamine H2 receptor, histamine H3 receptor, and histamine H4 receptor. Our hypothesis is that HRH-4 opens new possibility in treatment in allergy diseases and FXF could be the antagonist of both HRH-1 and HRH-4. We isolated a peripheral blood mononuclear cell (PBMC) from children with diagnosed allergies and healthy - control group and measured the HRH-1 and HRH-4 mRNA gene expression using Quantitive Real-Time PCR. We obtained the results from basal gene expression and after FXF and histamine stimulation. HRH-1 mRNA basal gene expression shows significantly higher, and HRH-4 shows significantly lower expression in allergy group compared to control. In both groups HRH-1 mRNA gene expression was observed as statistically significant increased after histamine stimulation compared to cells not treated, while in HRH-4 only in allergy group we observed statistical increase. FXF successively blocked histamine affinity in HRH-1 mRNA gene expression but not in HRH-4, where we not observed any reaction. Results clearly overturned our hypothesis about the possibility of using FXF to block over-expression HRH-4 and open new way of treatment in allergy diseases.

A Surface Plasmon Resonance Histamine Sensor Based on an Electropolymerized Molecularly Imprinted Polymer (E-MIP)

In this paper, histamine sensing by surface plasmon resonance (SPR) spectroscopy using an electropolymerized molecularly imprinted polymer (E-MIP) of functionalized polyterthiophene and histamine is reported. The MIP histamine sensor showed good linear relationship (R 2 = 0.995) between the SPR angle shift and the logarithm of histamine concentration, ranging from 15 – 500 μg/mL, with a limit of detection (LOD) of 2.0 μg/mL and a calibration sensitivity of 44.6 m° per log μg/mL histamine. Results also showed higher binding affinity of histamine for the MIP sensor than for the non-imprinted (NIP) sensor indicating excellent specificity. Moreover, the selectivity of the MIP histamine sensor against some of its structural analogues was demonstrated.

Asp73-dependent and -independent regulation of the affinity of ligands for human histamine H1 receptors by Na+

The affinity of ligands for G-protein-coupled receptors (GPCRs) is allosterically regulated by Na+ via a highly conserved aspartate residue (Asp2.50) in the second transmembrane domain of GPCRs. In the present study, we examined the Na+-mediated regulation of the affinity of ligands for Gq/11-protein-coupled human histamine H1 receptors in Chinese hamster ovary cells. The affinities of 3 agonists and 20 antihistamines were evaluated by their displacement curves against the binding of [3H]-mepyramine to membrane preparations in the presence or absence of 100mM NaCl. The affinities of most drugs including histamine, an agonist, and d-chlorpheniramine, a first-generation antihistamine, were reduced by NaCl, with the extent of NaCl-mediated changes varying widely between drugs. In contrast, the affinities of some second-generation antihistamines such as fexofenadine were increased by NaCl. These changes were retained in intact cells. The mutation of Asp2.50 (Asp73) to asparagine abrogated NaCl-induced reductions in affinities for histamine and d-chlorpheniramine, but not NaCl-induced increases in the affinity for fexofenadine. Quantitative structure-activity relationship (QSAR) analyses showed that these Na+-mediated changes were explained and predicted by a combination of the molecular energies and implicit solvation energies of the compounds. These results suggest that Na+ diversely regulates the affinity of ligands for H1 receptors from the extracellular sites of receptors via Asp73-dependent and -independent mechanisms in a manner that depends on the physicochemical properties of ligands. These results may contribute to a deeper understanding of the fundamental mechanisms by which the affinity of ligands for their receptors is allosterically regulated by Na+.

Leucine 208 in human histamine N-methyltransferase emerges as a hotspot for protein stability rationalizing the role of the L208P variant in intellectual disability

The degradation of histamine catalyzed by the SAM-dependent histamine N-methyltransferase (HNMT) is critically important for the maintenance of neurological processes. Recently, two mutations in the encoding human gene were reported to give rise to dysfunctional protein variants (G60D and L208P) leading to intellectual disability. In the present study, we have expressed eight L208 variants with either apolar (L208F and L208V), polar (L208N and L208T) or charged (L208D, L208H, L208K and L208R) amino acids to define the impact of side chain variations on protein structure and function. We found that the variants L208N, L208T, L208D and L208H were severely compromised in their stability. The other four variants were obtained in lower amounts in the order wild-type HNMT>L208F=L208V>L208K=L208R. Biochemical characterization of the two variants L208F and L208V exhibited similar Michaelis-Menten parameters for SAM and histamine while the enzymatic activity was reduced to 21% and 48%, respectively. A substantial loss of enzymatic activity and binding affinity for histamine was seen for the L208K and L208R variants. Similarly the thermal stability for the latter variants was reduced by 8 and 13°C, respectively. These findings demonstrate that position 208 is extremely sensitive to side chain variations and even conservative replacements affect enzymatic function. Molecular dynamics simulations showed that amino acid replacements in position 208 perturb the helical character and disrupt interactions with the adjacent β-strand, which is involved in the binding and correct positioning of histamine. This finding rationalizes the gradual loss of enzymatic activity observed in the L208 variants.

The Quantum Nature of Drug-Receptor Interactions: Deuteration Changes Binding Affinities for Histamine Receptor Ligands.

In this article we report a combined experimental and computational study concerning the effects of deuteration on the binding of histamine and two other histaminergic agonists to 3H-tiotidine-labeled histamine H2 receptor in neonatal rat astrocytes. Binding affinities were measured by displacing radiolabeled tiotidine from H2 receptor binding sites present on cultured neonatal rat astrocytes. Quantum-chemical calculations were performed by employing the empirical quantization of nuclear motion within a cluster model of the receptor binding site extracted from the homology model of the entire H2 receptor. Structure of H2 receptor built by homology modelling is attached in the supporting information (S1 Table) Experiments clearly demonstrate that deuteration affects the binding by increasing the affinity for histamine and reducing it for 2-methylhistamine, while basically leaving it unchanged for 4-methylhistamine. Ab initio quantum-chemical calculations on the cluster system extracted from the homology H2 model along with the implicit quantization of the acidic N–H and O–H bonds demonstrate that these changes in the binding can be rationalized by the altered strength of the hydrogen bonding upon deuteration known as the Ubbelohde effect. Our computational analysis also reveals a new mechanism of histamine binding, which underlines an important role of Tyr250 residue. The present work is, to our best knowledge, the first study of nuclear quantum effects on ligand receptor binding. The ligand H/D substitution is relevant for therapy in the context of perdeuterated and thus more stable drugs that are expected to enter therapeutic practice in the near future. Moreover, presented approach may contribute towards understanding receptor activation, while a distant goal remains in silico discrimination between agonists and antagonists based on the receptor structure.

Evolution of histamine oxidase activity for biotechnological applications

Histamine is present to various degrees in many foods, and concentrations in fish samples are considered a good indicator of freshness and hygienic food quality. Seeking for innovative methods to quantify histamine in foods, we used a synthetic gene designed on the sequence of histamine oxidase from Arthrobacter crystallopoietes (HOD) as the starting point in this study to develop a biosensor. HOD was expressed in Escherichia coli cells with a yield of ∼7mg protein/L of fermentation broth. Recombinant wild-type HOD oxidized histamine and tyramine whereas it was inactive toward putrescine and cadaverine (two amines present in fish samples). The putative residues involved in substrate binding were identified by an in silico docking procedure based on a model of the structure of HOD: site-saturation mutagenesis was performed on 8 positions. The most significant changes in kinetic properties were observed for the P143M HOD: this variant showed higher histamine affinity and lower substrate inhibition by tyramine than wild-type enzyme. Biosensor prototypes were produced using both the wild-type and the P143M variant HOD. These biosensors showed a good sensitivity and selectivity with respect to biogenic amines present in food specimens. Accordingly, the HOD-based biosensor was successfully used to assess histamine in fish samples, yielding values in good agreement with those obtained by HPLC analyses but in a few seconds and at a significantly lower cost per analysis.

Preparation and Evaluation of Histamine Imprinted Polymer as a Selective Sorbent in Molecularly Imprinted Solid-Phase Extraction Coupled with High Performance Liquid Chromatography Analysis in Canned Fish

A series of molecularly imprinted polymers (MIPs) were prepared against histamine. Different template/monomer ratios were applied to optimize the imprinting condition. Methacrylic acid (MAA) as a functional monomer and Chloroform as a solvent were applied in polymerization process. The binding properties of MIPs were studied in comparison with a blank non-imprinted polymer. The optimized polymer, with a histamine/MAA ratio of 1/4, was selected as a sorbent in molecularly imprinted solid-phase extraction (MISPE) of histamine from canned fish. Scatchard analysis of MIP-histamine interactions revealed two types of binding sites for MIP: high affinity (KD = 11.11 μM) and low affinity (KD = 333.3 μM). The MISPE procedure was calibrated and a recovery of 76.5–97.6 % was obtained. The intra-and inter-day precision values were less than 5.70 % and 10.1 %, respectively. The selectivity of MISPE for histamine was also studied in comparison with some other structurally similar amines, which could be simultaneously present in canned fish. The performance of the imprinted polymer was examined and the results indicated that its good selectivity and affinity for histamine was very promising. Therefore, the proposed calibrated method could be applied in selective extraction and analysis of histamine in canned fish.

Molecularly imprinted polymers for histamine recognition in aqueous environment

Molecularly imprinted polymers (MIP) for histamine using methacrylic acid were developed and recognition mechanisms were thoroughly characterized for the first time in this study. The binding affinity of imprinted polymer with structurally related compounds was studied in organic and aqueous media, at various conditions. In organic media, MIP was found to bind histamine two and six times more than ranitidine and fluoxetine, respectively, whereas higher selectivity was observed in the case of dimentidene or disodium cromoglycate. The specific binding sites of MIP recognized histamine over L-histidine in aqueous conditions, while higher affinity for histamine compared to ranitidine, disodium cromoglycate, putrescine and to a putrescine analogue was observed. A combination of NMR and UV spectroscopy analyses for investigation of imprinting and recognition properties revealed that strong specific interactions between the functional monomer and histamine in the prepolymerization and in the aqueous solutions were probably responsible for histamine recognition. The preparation of histamine MIPs and elucidation of imprinting and recognition mechanism may serve as useful insight for future application of MIPs.

The Affinity of Histamine for Gq Protein-Coupled Histamine H1-Receptors Is Predominantly Regulated by Their Internalization in Human Astrocytoma Cells

We examined the regulatory mechanisms of the affinity of Gq protein-coupled histamine H1-receptors for histamine after histamine pretreatment in intact human U373 MG astrocytoma cells. In control cells, the displacement curves for histamine against the binding of 5 nM [3H] mepyramine, a radioligand for H1-receptors, showed the presence of two binding sites for histamine, that is, high and low affinity sites. Pretreatment with 0.1 mM histamine for 30 min at 37°C induced a significant reduction in the percentage of high affinity sites for histamine and a concomitant increase in the percentage of low affinity sites with no change in their pIC50 values. These histamine-induced changes were insensitive to 30 μM KN-62, an inhibitor of Ca2+/calmodulin-dependent protein kinase II, but they were completely inhibited either by 0.4 mM ZnCl2, an inhibitor of G protein-coupled receptor kinases (GRKs), or under hypertonic conditions, where clathrin-mediated endocytosis is known to be inhibited. These results suggest that histamine-induced conversion of high to low affinity sites for histamine is predominantly regulated by GRK/clathrin-mediated internalization of H1-receptors in human astrocytoma cells.

Atypical antipsychotics and the neural regulation of food intake and peripheral metabolism

The atypical antipsychotics (AAPs) are associated with weight gain and an increased incidence of metabolic disease including type 2 diabetes mellitus. Epidemiological, cross-sectional and prospective studies suggest that two of the AAPs, olanzapine and clozapine, cause the most dramatic weight gain and metabolic impairments including increased fasting glucose, insulin and triglycerides. Relative to the other AAPs, both olanzapine and clozapine exhibit a particularly high antagonistic affinity for histamine and muscarinic receptors which have been hypothesized as mediators of the reported increase in weight and glucose abnormalities. In this article, we review the current evidence for the AAP associated weight gain and abnormal glucose metabolism. We postulate that the effects of the AAPs on food intake and peripheral metabolism are initially independently regulated but with increasing body adiposity, the early AAP-induced impairments in peripheral metabolism will be exacerbated, thereby establishing a vicious cycle such that the effects of the AAP are magnified by the known pathophysiological consequences of obesity. Furthermore, we examine how inhibition of the histaminergic pathway may mediate increases in food intake and the potential role of the vagus nerve in the reported peripheral metabolic effects.

Expression and functional properties of canine, rat, and murine histamine H4 receptors in Sf9 insect cells

The histamine H₄ receptor (H₄R) is expressed on cells of the immune system including eosinophils, dendritic cells, and T cells and plays an important role in the pathogenesis of bronchial asthma, atopic dermatitis, and pruritus. Analysis of the H₄R in these diseases depends on the use of animal models. However, there are substantial pharmacological differences between various H₄R species orthologs. The purpose of this study was to analyze the pharmacological properties of canine, rat, and murine H₄R in comparison to human H₄R expressed in Sf9 insect cells. Only hH₄R and cH₄R exhibited a sufficiently high [³H]histamine affinity for radioligand binding studies. Generally, cH₄R exhibited lower ligand-affinities than hH₄R. Similarly, in high-affinity GTPase studies, ligands were more potent at hH₄R than at other H₄R species orthologs. Unlike the other H₄R species orthologs, hH₄R exhibited high agonist-independent (constitutive) activity. Most strikingly, the prototypical H₄R antagonist (1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine) (JNJ7777120) exhibited partial agonistic activity at cH₄R, rH₄R, and mH₄R, whereas at hH₄R, JNJ7777120 was a partial inverse agonist. H₄R agonists from the class of N ( G )-acylated imidazolylpropylguanidines and cyanoguanidines exhibited substantial differences in terms of affinity, potency, and efficacy among H₄R species orthologs, too. The species-dependent pharmacological profiles are not due to the highly variable amino acid sequence position 341. Finally, H₄R species orthologs differ from each other in terms of regulation by NaCl. Collectively, there are profound pharmacological differences between H₄R species orthologs. Most importantly, caution must be exerted when interpreting pharmacological effects of "the prototypical H₄R antagonist" JNJ7777120 as H₄R antagonism.